Lipocalin-2 Deficiency Diminishes Canonical NLRP3 Inflammasome Formation and IL-1ß Production in the Subacute Phase of Spinal Cord Injury.

Spinal cord injury (SCI) results in the production of proinflammatory cytokines due to inflammasome activation. Lipocalin 2 (LCN2) is a small secretory glycoprotein upregulated by toll-like receptor (TLR) signaling in various cells and tissues. LCN2 secretion is induced by infection, injury, and metabolic disorders. In contrast, LCN2 has been implicated as an anti-inflammatory regulator. However, the role of LCN2 in inflammasome activation during SCI remains unknown. This study examined the role of Lcn2 deficiency in the NLRP3 inflammasome-dependent neuroinflammation in SCI. Lcn2-/- and wild-type (WT) mice were subjected to SCI, and locomotor function, formation of the inflammasome complex, and neuroinflammation were assessed. Our findings demonstrated that significant activation of the HMGB1/PYCARD/caspase-1 inflammatory axis was accompanied by the overexpression of LCN2 7 days after SCI in WT mice. This signal transduction results in the cleaving of the pyroptosis-inducing protein gasdermin D (GSDMD) and the maturation of the proinflammatory cytokine IL-1ß. Furthermore, Lcn2-/- mice showed considerable downregulation in the HMGB1/NLRP3/PYCARD/caspase-1 axis, IL-1ß production, pore formation, and improved locomotor function compared with WT. Our data suggest that LCN2 may play a role as a putative molecule for the induction of inflammasome-related neuroinflammation in SCI.

Lipocalin 2 attenuates oligodendrocyte loss and immune cell infiltration in mouse models for multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system disease characterized by both degenerative and inflammatory processes. Various mediators are involved in the interplay of degeneration and innate immunity on one hand and peripheral adaptive immunity on the other hand. The secreted protein lipocalin 2 (LCN2) is an inflammatory modulator in a variety of pathologies. Although elevated intrathecal levels of LCN2 have been reported in MS patients, it's functional role is widely unknown. Here, we identified a subpopulation of astrocytes as a source of LCN2 in MS lesions and respective animal models. We investigated the functional role of LCN2 for both autoimmune and degenerative aspects in three MS mouse models including both wild type (WT) and Lcn2-/- mouse strains. While the experimental autoimmune encephalomyelitis (EAE) model reflects primary autoimmunity, the cuprizone model reflects selective oligodendrocyte loss and demyelination. In addition, we included a combinatory Cup/EAE model in which primary cytodegeneration is followed by inflammatory lesions within the forebrain. While in the EAE model, the disease outcome was comparable in between the two mouse strains, cuprizone intoxicated Lcn2-/- animals showed an increased loss of oligodendrocytes. In the Cup/EAE model, Lcn2-/- animals showed increased inflammation when compared to WT mice. Together, our results highlight LCN2 as a potentially protective molecule in MS lesion formation, which might be able to limit loss of oligodendrocytes immune-cell invasion. Despite these findings, it is not yet clear which glial cell phenotype (and to which extent) contributes to the observed neuroprotective effects, that is, microglia and/or astroglia or even endothelial cells in the brain.

Astrocytic Nrf2 expression protects spinal cord from oxidative stress following spinal cord injury in a male mouse model.

BACKGROUND: Spinal cord injury (SCI) induces a multitude of deleterious processes, including neuroinflammation and oxidative stress (OS) which contributed to neuronal damage and demyelination. Recent studies have suggested that increased formation of reactive oxygen species (ROS) and the consequent OS are critical events associated with SCI. However, there is still little information regarding the impact of these events on SCI. Astrocytes are key regulators of oxidative homeostasis in the CNS and astrocytic antioxidant responses promote the clearance of oxidants produced by neurons. Therefore, dysregulation of astrocyte physiology might largely contribute to oxidative damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of cellular anti-oxidative stress responses. METHODS: In the current study, we hypothesized that astrocytic activation of Nrf2 protects the spinal cord post injury via suppression of neuroinflammation. Thus, using mice line with a GFAP-specific kelch-like ECH-associated protein 1 (Keap1)-deletion, we induced a hyperactivation of Nrf2 in astrocytes and further its effects on SCI outcomes. SCI-induction was performed in mice using the Infinite Horizon Spinal Cord Impactor with a force of 60 kdyn. To assess the quantitative pattern of Nrf2/ARE-activation, we included transgenic ARE-Luc mice. Data were analyzed with GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Brown-Forsythe test was performed to test for equal variances and normal distribution was tested with Shapiro-Wilk. RESULTS: In ARE-Luc mice, a significant induction of luciferase-activity was observed as early as 1 day post-injury, indicating a functional role of Nrf2-activity at the epicenter of SCI. Furthermore, SCI induced loss of neurons and oligodendrocytes, demyelination and inflammation in wild type mice. The loss of myelin and oligodendrocytes was clearly reduced in Keap1 KO mice. In addition, Keap-1 KO mice showed a significantly better locomotor function and lower neuroinflammation responses compared to wild type mice. CONCLUSIONS: In summary, our in vivo bioluminescence data showed Nrf2-ARE activation during primary phase of SCI. Furthermore, we found that cell specific hyperactivation of Nrf2 was sufficient to protect the spinal cord against injury which indicate a promising therapeutic approach for SCI-treatment.

Hypoxia Induces Astrocyte-Derived Lipocalin-2 in Ischemic Stroke.

Ischemic stroke causes rapid hypoxic damage to the core neural tissue which is followed by graded chronological tissue degeneration in the peri-infarct zone. The latter process is mainly triggered by neuroinflammation, activation of inflammasomes, proinflammatory cytokines, and pyroptosis. Besides microglia, astrocytes play an important role in the fine-tuning of the inflammatory network in the brain. Lipocalin-2 (LCN2) is involved in the control of innate immune responses, regulation of excess iron, and reactive oxygen production. In this study, we analyzed LCN2 expression in hypoxic rat brain tissue after ischemic stroke and in astrocyte cell cultures receiving standardized hypoxic treatment. Whereas no LCN2-positive cells were seen in sham animals, the number of LCN2-positive cells (mainly astrocytes) was significantly increased after stroke. In vitro studies with hypoxic cultured astroglia revealed that LCN2 expression is significantly increased after only 2 h, then further increased, followed by a stepwise decline. The expression pattern of several proinflammatory cytokines mainly followed that profile in wild type (WT) but not in cultured LCN2-deficient astrocytes. Our data revealed that astrocytes are an important source of LCN2 in the peri-infarct region under hypoxic conditions. However, we must also stress that brain-intrinsic LCN2 after the initial hypoxia period might come from other sources such as invaded immune cells and peripheral organs via blood circulation. In any case, secreted LCN2 might have an influence on peripheral organ functions and the innate immune system during brain hypoxia.

Lipocalin 2 receptors: facts, fictions, and myths.

The human 25-kDa Lipocalin 2 (LCN2) was first identified and purified as a protein that in part is associated with gelatinase from neutrophils. This protein shows a high degree of sequence similarity with the deduced sequences of rat α2-microglobulin-related protein and the mouse protein 24p3. Based on its typical lipocalin fold, which consists of an eight-stranded, anti-parallel, symmetrical ß-barrel fold structure it was initially thought that LCN2 is a circulating protein functioning as a transporter of small lipophilic molecules. However, studies in Lcn2 null mice have shown that LCN2 has bacteriostatic properties and plays a key role in innate immunity by sequestering bacterial iron siderophores. Numerous reports have further shown that LCN2 is involved in the control of cell differentiation, energy expenditure, cell death, chemotaxis, cell migration, and many other biological processes. In addition, important roles for LCN2 in health and disease have been identified in Lcn2 null mice and multiple molecular pathways required for regulation of Lcn2 expression have been identified. Nevertheless, although six putative receptors for LCN2 have been proposed, there is a fundamental lack in understanding of how these cell-surface receptors transmit and amplify LCN2 to the cell. In the present review we summarize the current knowledge on LCN2 receptors and discuss inconsistencies, misinterpretations and false assumptions in the understanding of these potential LCN2 receptors.

Neuroprotective effect of the Nrf2/ARE/miRNA145-5p signaling pathway in the early phase of spinal cord injury.

AIMS: Spinal cord injury (SCI) is a debilitating neurological condition often associated with chronic neuroinflammation and redox imbalance. Oxidative stress is one of the main hallmark of secondary injury of SCI which is tightly regulated by nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) signaling. In this study, we aimed at investigating the interplay between inflammation-related miRNAs and the Nrf2 pathway in animal model of SCI. MATERIALS AND METHODS: The expression of selected four validated miRNA-target pairs (miRNA223-3p, miRNA155-5p, miRNA145-5p, and miRNA124-3p) was examined at different time points (6 h, 12 h, 1 day, 3 day and 7 day) after SCI. Further, using GFAP-specific kelch-like ECH-associated protein 1 deletion (Keap1-/-) and whole-body Nrf2-/- knockout mice, we investigated the potential interplay between each miRNA and the Keap1/Nrf2 signaling system. KEY FINDINGS: The expression of all miRNAs except miRNA155-5p significantly increased 24 h after SCI and decreased after 7 days. Interestingly, Keap1-/- mice only showed significant increase in the miRNA145-5p after 24 h SCI compared to the WT group. In addition, Keap1-/- mice showed significant decrease in CXCL10/12 (CXCL12 increased in Nrf2-/- mice), and TNF-α, and an increase in Mn-SOD and NQO-1 (Mn-SOD and NQO-1 decreased in Nrf2-/- mice) compared to WT mice. SIGNIFICANCE: Our results suggest that astrocytic hyperactivation of Nrf2 exert neuroprotective effects at least in part through the upregulation of miRNA145-5p, a negative regulator of astrocyte proliferation, and induction of ARE in early phase of SCI. Further studies are needed to investigate the potential interplay between Nrf2 and miRNA145-5p in neuroinflammatory condition.

Long-Term Glucose Starvation Induces Inflammatory Responses and Phenotype Switch in Primary Cortical Rat Astrocytes.

Astrocytes are the most abundant cell type in the brain and crucial to ensure the metabolic supply of neurons and their synapse formation. Overnutrition as present in patients suffering from obesity causes astrogliosis in the hypothalamus. Other diseases accompanied by malnutrition appear to have an impact on the brain and astrocyte function. In the eating disorder anorexia nervosa (AN), patients suffer from undernutrition and develop volume reductions of the cerebral cortex, associated with reduced astrocyte proliferation and cell count. Although an effect on astrocytes and their function has already been shown for overnutrition, their role in long-term undernutrition remains unclear. The present study used primary rat cerebral cortex astrocytes to investigate their response to chronic glucose starvation. Cells were grown with a medium containing a reduced glucose concentration (2 mM) for 15 days. Long-term glucose starvation increased the expression of a subset of pro-inflammatory genes and shifted the primary astrocyte population to the pro-inflammatory A1-like phenotype. Moreover, genes encoding for proteins involved in the unfolded protein response were elevated. Our findings demonstrate that astrocytes under chronic glucose starvation respond with an inflammatory reaction. With respect to the multiple functions of astrocytes, an association between elevated inflammatory responses due to chronic starvation and alterations found in the brain of patients suffering from undernutrition seems possible.

Lipocalin 2 as a Putative Modulator of Local Inflammatory Processes in the Spinal Cord and Component of Organ Cross talk After Spinal Cord Injury.

Lipocalin 2 (LCN2), an immunomodulator, regulates various cellular processes such as iron transport and defense against bacterial infection. Under pathological conditions, LCN2 promotes neuroinflammation via the recruitment and activation of immune cells and glia, particularly microglia and astrocytes. Although it seems to have a negative influence on the functional outcome in spinal cord injury (SCI), the extent of its involvement in SCI and the underlying mechanisms are not yet fully known. In this study, using a SCI contusion mouse model, we first investigated the expression pattern of Lcn2 in different parts of the CNS (spinal cord and brain) and in the liver and its concentration in blood serum. Interestingly, we could note a significant increase in LCN2 throughout the whole spinal cord, in the brain, liver, and blood serum. This demonstrates the diversity of its possible sites of action in SCI. Furthermore, genetic deficiency of Lcn2 (Lcn2-/-) significantly reduced certain aspects of gliosis in the SCI-mice. Taken together, our studies provide first valuable hints, suggesting that LCN2 is involved in the local and systemic effects post SCI, and might modulate the impairment of different peripheral organs after injury.

Inflammatory Responses of Astrocytes Are Independent from Lipocalin 2.

The central nervous system (CNS) responds to diverse neurologic injuries with a vigorous activation of astrocytes. In addition to their role in the maintenance of CNS homeostasis and neuronal function, astrocytes are thought to participate in the regulation of innate and adaptive immune responses in the CNS. Following antigen recognition, reactive astrocytes may participate in the initiation of innate immune responses, and modulate adaptive immune response leading to the recruitment of peripheral immune cells. Among activation, astrocytes undergo morphological changes and express several molecules, e.g., chemokines. Lipocalin 2 (LCN2) is involved in the control of innate immune responses, regulation of excess iron, and reactive oxygen production. Here, we investigated the influence of LCN2 on basic astrocytic functions linked to inflammatory responses. In vitro studies revealed a similar chemokine expression pattern in wild-type and Lcn2-deficient astrocyte cultures after treatment with lipopolysaccharides (LPS). Increased wound closure and morphological changes upon LPS treatment are independent of Lcn2 expression. We conclude that LCN2 is not necessary for basic astrocytic functions in the context of inflammation. However, CNS-derived LCN2 might have a regulatory effect on other cells, e.g., endothelial cells of the blood-brain barrier.